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| United States Patent |
6,232,441
|
|
Wu
, et al.
|
May 15, 2001
|
PIGR-1, a member of immunoglobulin gene superfamily
Abstract
PIGR-1 polypeptides and polynucleotides and methods for producing such
polypeptides by recombinant techniques are disclosed. Also disclosed are
methods for utilizing PIGR-1 polypeptides and polynucleotides in the
design of protocols for the treatment of rheumatoid arthritis (RA),
multiple sclerosis (MS), psoriasis, systemic lupus erythematosus (SLE) and
inflammatory bowel disease (IBD), among others and diagnostic assays for
such conditions.
| Inventors:
|
Wu; Shujian (Levittown, PA);
Sweet; Raymond W (Bala Cynwyd, PA);
Truneh; Alemseged (West Chester, PA);
Hurle; Mark Robert (Norristown, PA)
|
| Assignee:
|
SmithKline Beecham Corporation (Philadelphia, PA)
|
| Appl. No.:
|
300985 |
| Filed:
|
April 28, 1999 |
| Current U.S. Class: |
530/350; 530/395 |
| Intern'l Class: |
C07K 014/435 |
| Field of Search: |
530/350,395
|
References Cited
Other References
Jackson, David G., et al., "Molecular cloning of a novel member of the
immunoglobulin gene superfamily homologous to the polymeric immunoglobulin
receptor", Eur. J. Immunol. 22:1157-1163, (1992).
Danish, A., et al., "Expression of the CMRF-35 antigen, a new member of the
immunoglobulin gene superfamily, is differentially regulated on
leucocytes", Immunology 79:55-63, (1993).
Krajci et al. "Molecular Cloning Of The Human Transmembrane Secretory
Component (Poly-Ig Receptor) And Its mRNA Expression In Human Tissues",
Biochemical and Biophysical Research Communications, vol. 158 (3), pp.
783-789 (1989).
Kariv et al. "Analysis of the Site of Interaction of CD28 with Its
Counter-Receptors CD80 and CD86 And Correlation with Function", Journal of
Immunology, vol. 157 (1), pp. 29-38 (1996).
GenBank Accession No. X66171. (Aug. 2, 1993).
Banting, G., et al., Intracellular targetting signals of polymeric
immunoglobulin receptors are highly conserved between species. FEBS Let.
254(1,2):177-183, Aug. 1989.*
Cardone, M.H., et al., Signal transduction by the polymeric immunoglobulin
receptor suggest a role in regulation of receptor transcytosis. J. Cell
Biol. 133(5):997-1005, Jun. 1996.
|
Primary Examiner: Saunders; David
Assistant Examiner: Tung; Mary Beth
Attorney, Agent or Firm: Han; William T.
Ratner & Prestia, King; William T.
Parent Case Text
This application is a division of U.S. application Ser. No. 08/955,937,
filed Oct. 22, 1997, now U.S. Pat. No. 6,020,161. which claims the benefit
of U.S. Provisional Application No. 60/056,152, filed Aug. 19, 1997 the
entire contents of which are incorporated herein by reference.
Claims
What is claimed is:
1. An isolated PIGR-1 polypeptide comprising the amino acid sequence of SEQ
ID NO:2.
2. The isolated polypeptide of claim 1 consisting of the amino acid
sequence of SEQ ID NO:2.
Description
FIELD OF INVENTION
This invention relates to newly identified polynucleotides, polypeptides
encoded by them and to the use of such polynucleotides and polypeptides,
and to their production. More particularly, the polynucleotides and
polypeptides of the present invention relate to Immunoglobulin
superfamily, hereinafter referred to as PIGR-1. The invention also relates
to inhibiting or activating the action of such polynucleotides and
polypeptides.
BACKGROUND OF THE INVENTION
The immunoglobulin (Ig) gene superfamily comprises a large number of cell
surface glycoproteins that share sequence homology with the V and C
domians of antibody heavy and light chains. These molecules function as
receptors for antigen, immunoglobulin and cytokines as well as adhesion
molecules (A. F. Williams et al., Annu. Rev. Immunol. 6:381-405, 1988).
Most Ig superfamily members are relatively complex polydomain molecules
cotaining multiple Ig V- and C-like domains (T. Hunkapiller et al., Adv.
Immunol. 44:1-63, 1989). However, a subset of them have relatively simple
structures containing only a single Ig domain in the extracellular region.
Examples of this type of receptors are CD28 and CD8 (A. Aruffo et al.,
Proc. Natl. Acad. Sci. USA 84:8573-8577, 1987) Recently, CMRF-35, an novel
membrane glycoprotein of the Ig gene superfamily containing a single
extracellular Ig V domain, was identified by D. G. Jackson et al., Eur. J.
Immunol. 22:1157-1163, 1992. CMRF-35 is exclusively detected on cells from
both the myeloid and lymphoid differentiation pathways. However,
expression of this gene is markedly influenced by stimulation of
leucocytes with mitogens and cytokines (A. Daish et al., Immunology
79:55-63, 1993). This suggests that CMRF-35 may be strongly associated
with differentiation and proliferation of diverse leucocytes types. This
indicates that these receptors have an established, proven history as
therapeutic targets. Clearly there is a need for identification and
characterization of further receptors which can play a role in preventing,
ameliorating or correcting dysfunctions or diseases, including, but not
limited to, rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis,
systemic lupus erythematosus (SLE) and Inflammatory Bowel Disease (IBD).
SUMMARY OF THE INVENTION
In one aspect, the invention relates to PIGR-1 polypeptides and recombinant
materials and methods for their production. Another aspect of the
invention relates to methods for using such PIGR-1 polypeptides and
polynucleotides. Such uses include the treatment of rheumatoid arthritis
(RA), multiple sclerosis (MS), psoriasis, systemic lupus erythematosus
(SLE) and inflammatory bowel disease (IBD), among others. In still another
aspect, the invention relates to methods to identify agonists and
antagonists using the materials provided by the invention, and treating
conditions associated with PIGR-1 imbalance with the identified compounds.
Yet another aspect of the invention relates to diagnostic assays or
detecting diseases associated with inappropriate PIGR-1 activity or
levels.
DESCRIPTION OF THE INVENTION
Definitions
The following definitions are provided to facilitate understanding of
certain terms used frequently herein.
"PIGR-1" refers, among others, to a polypeptide comprising the amino acid
sequence set forth in SEQ ID NO:2, or an allelic variant thereof.
"Receptor Activity" or "Biological Activity of the Receptor" refers to the
metabolic or physiologic function of said PIGR-1 including similar
activities or improved activities or these activities with decreased
undesirable side-effects. Also included are antigenic and immunogenic
activities of said PIGR-1.
"PIGR-1 gene" refers to a polynucleotide comprising the nucleotide sequence
set forth in SEQ ID NO:1 or allelic variants thereof and/or their
complements.
"Antibodies" as used herein includes polyclonal and monoclonal antibodies,
chimeric, single chain, and humanized antibodies, as well as Fab
fragments, including the products of an Fab or other immunoglobulin
expression library.
"Isolated" means altered "by the hand of man" from the natural state. If an
"isolated" composition or substance occurs in nature, it has been changed
or removed from its original environment, or both. For example, a
polynucleotide or a polypeptide naturally present in a living animal is
not "isolated," but the same polynucleotide or polypeptide separated from
the coexisting materials of its natural state is "isolated", as the term
is employed herein.
"Polynucleotide" generally refers to any polyribonucleotide or
polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA
or DNA. "Polynucleotides" include, without limitation single- and
double-stranded DNA, DNA that is a mixture of single- and double-stranded
regions, single- and double-stranded RNA, and RNA that is mixture of
single- and double-stranded regions, hybrid molecules comprising DNA and
RNA that may be single-stranded or, more typically, double-stranded or a
mixture of single- and double-stranded regions. In addition,
"polynucleotide" refers to triple-stranded regions comprising RNA or DNA
or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs
containing one or more modified bases and DNAs or RNAs with backbones
modified for stability or for other reasons. "Modified" bases include, for
example, tritylated bases and unusual bases such as inosine. A variety of
modifications has been made to DNA and RNA; thus, "polynucleotide"
embraces chemically, enzymatically or metabolically modified forms of
polynucleotides as typically found in nature, as well as the chemical
forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide"
also embraces relatively short polynucleotides, often referred to as
oligonucleotides.
"Polypeptide" refers to any peptide or protein comprising two or more amino
acids joined to each other by peptide bonds or modified peptide bonds,
i.e., peptide isosteres. "Polypeptide" refers to both short chains,
commonly referred to as peptides, oligopeptides or oligomers, and to
longer chains, generally referred to as proteins. Polypeptides may contain
amino acids other than the 20 gene-encoded amino acids. "Polypeptides"
include amino acid sequences modified either by natural processes, such as
posttranslational processing, or by chemical modification techniques which
are well known in the art. Such modifications are well described in basic
texts and in more detailed monographs, as well as in a voluminous research
literature. Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or carboxyl
termini. It will be appreciated that the same type of modification may be
present in the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched as a result of ubiquitination,
and they may be cyclic, with or without branching. Cyclic, branched and
branched cyclic polypeptides may result from posttranslation natural
processes or may be made by synthetic methods. Modifications include
acetylation, acylation, ADP-ribosylation, amidation, covalent attachment
of flavin, covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid or
lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation, demethylation,
formation of covalent cross-links, formation of cystine, formation of
pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proeolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated addition of
amino acids to proteins such as arginylation, and ubiquitination. See, for
instance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F.,
Posttranslational Protein Modifications: Perspectives and Prospects, pgs.
1-12 in POSTTRANSLATION COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,
Ed., Academic Press, Now York, 1983; Seifter et al., "Analysis for protein
modifications and nonprotein cofactors", Meth Enzymol (1990) 182:626-646
and Rattan et al., "Protein Synthesis: Posttranslational Modifications and
Aging", Ann NY Acad Sci (1992) 663:48-62.
"Variant" as the term is used herein, is a polynucleotide or polypeptide
that differs from a reference polynucleotide or polypeptide respectively,
but retains essential properties. A typical variant of a polynucleotide
differs in nucleotide sequence from another, referenced polynucleotide.
Changes in the nucleotide sequence of the variant may or may not alter the
amino acid sequence of a polypeptide encoded by the reference
polynucleotide. Nucleotide changes may result in amino acids
substitutions, additions, deflections, fusions and truncations in the
polypeptide encoded by the reference sequence, as discussed below. A
typical variant of a polypeptide differs in amino acid sequence from
another, reference polypeptide. Generally, differences are limited so that
the sequences of the reference polypeptide and the variant are closely
overall and, in many regions, identical. A variant and reference
polypeptide may differ in amino acid sequence by one or more
substitutions, additions, deletions in any combination. A substituted or
inserted amino acid residue may or may not be one encoded by the genetic
code. A variant of a polynucleotide or polypeptide may be a naturally
occurring such as an allelic variant, or it may be a variant that is not
known to occur naturally. Non-naturally occurring variants of
polynucleotides and polypeptides may be made by mutagenesis techniques or
by direct synthesis.
"Identity" is a measure of the identity of nucleotide sequences or amino
acid sequences. In general, the sequences are aligned so that the highest
order match is obtained. "Identity" per se has an art-recognized meaning
and can be calculated using published techniques. See, e.g.:
(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University
Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS,
Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF
SEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., Humana
Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von
Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there
exist a number of methods to measure identity between two polynucleotide
or polypeptide sequences, the term "identity" is well known to skilled
artisans (Carillo, H., and Lipton, D., SIAM J Applied Math (1988)
48:1073). Methods commonly employed to determine identity or similarity
between two sequences include, but are not limited to, those disclosed in
Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego,
1994, and Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073.
Methods to determine identity and similarity are codified in computer
programs. Preferred computer program methods to determine identity and
similarity between two sequences include, but are not limited to, GCS
program package (Devereux, J., et al., Nucleic Acids Research (1984)
12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J Molec Biol
(1990) 215:403).
As an illustration, by a polynucleotide having a nucleotide sequence having
at least, for example, 95% "identity" to a reference nucleotide sequence
of SEQ ID NO: 1 is intended that the nucleotide sequence of the
polynucleotide is identical to the reference sequence except that the
polynucleotide sequence may include up to five point mutations per each
100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1. In
other words, to obtain a polynucleotide having a nucleotide sequence at
least 95% identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted with
another nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the reference sequence may be inserted into the reference
sequence. These mutations of the reference sequence may occur at the 5 or
3 terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually among
nucleotides in the reference sequence or in one or more contiguous groups
within the reference sequence.
Similarly, by a polypeptide having an amino acid sequence having at least,
for example, 95% "identity" to a reference amino acid sequence of SEQ ID
NO:2 is intended that the amino acid sequence of the polypeptide is
identical to the reference sequence except that the polypeptide sequence
may include up to five amino acid alterations per each 100 amino acids of
the reference amino acid of SEQ ID NO: 2. In other words, to obtain a
polypeptide having an amino acid sequence at least 95% identical to a
reference amino acid sequence, up to 5% of the amino acid residues in the
reference sequence may be deleted or substituted with another amino acid,
or a number of amino acids up to 5% of the total amino acid residues in
the reference sequence may be inserted into the reference sequence. These
alterations of the reference sequence may occur at the amino or carboxy
terminal positions of the reference amino acid sequence or anywhere
between those terminal positions, interspersed either individually among
residues in the reference sequence or in one or more contiguous groups
within the reference sequence.
Polypeptides of the Invention
In one aspect, the present invention relates to PIGR-1 polypeptides (or
PIGR-1 proteins). The PIGR-1 polypeptides include the polypeptides of SEQ
ID NOS:2 and 4; as well as polypeptides comprising the amino acid sequence
of SEQ ID NO:2; and polypeptides comprising the amino acid sequence which
have at least 80% identity to that of SEQ ID NO:2 over its entire length,
and still more preferably at least 90% identity, and even still more
preferably at least 95% identity to SEQ ID NO: 2. Furthermore, those with
at least 97-99% are highly preferred. Also included within PIGR-1
polypeptides are polypeptides having the amino acid sequence which have at
least 80% identity to the polypeptide having the amino acid sequence of
SEQ ID NO: 2 over its entire length, and still more preferably at least
90% identity, and even still more preferably at least 95% identity to SEQ
ID NO: 2. Furthermore, those with at least 97-99% are highly preferred.
Preferably PIGR-1 polypeptides exhibit at least one biological activity of
the receptor.
The PIGR-1 polypeptides may be in the form of the "mature" protein or may
be a part of a larger protein such as a fusion protein. It is often
advantageous to include an additional amino acid sequence which contains
secretory or leader sequences, pro-sequences, sequences which aid in
purification such as multiple histidine residues, or an additional
sequence for stability during recombinant production.
Fragments of the PIGR-1 polypeptides are also included in the invention. A
fragment is a polypeptide having an amino acid sequence that entirely is
the same as part, but not all, of the amino acid sequence of the
aforementioned PIGR-1 polypeptides. As with PIGR-1 polypeptides, fragments
may be "free standing," or comprised within a larger polypeptide of which
they form a part or region, most preferably as a single continuous region.
Representative examples of polypeptide fragments of the invention,
include, for example, fragments from about amino acid number 1-20, 21-40,
41-60, 61-80, 81-100, and 101 to the end of PIGR-1 polypeptide. In this
context "about" includes the particularly recited ranges larger or smaller
by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both
extremes.
Preferred fragments include, for example, truncation polypeptides having
the amino acid sequence of PIGR-1 polypeptides, except for deletion of a
continuous series of residues that includes the amino terminus, or a
continuous series of residues that includes the carboxyl terminus or
deletion of two continuous series of residues, one including the amino
terminus and one including the carboxyl terminus. Also preferred are
fragments characterized by structural or functional attributes such as
fragments that comprise alpha-helix and alpha-helix forming regions,
beta-sheet and beta-sheet-forming regions, turn and turn-forming regions,
coil and coil-forming regions, hydrophilic regions, hydrophobic regions,
alpha amphipathic regions, beta amphipathic regions, flexible regions,
surface-forming regions, substrate binding region, and high antigenic
index regions. Other preferred fragments are biologically active
fragments. Biologically active fragments are those that mediate receptor
activity, including those with a similar activity or an improved activity,
or with a decreased undesirable activity. Also included are those that are
antigenic or immunogenic in an animal, especially in a human.
Preferably, all of these polypeptide fragments retain the biological
activity of the receptor, including antigenic activity. Among the most
preferred fragment is that having the amino acid sequence of SEQ ID NO: 4.
Variants of the defined sequence and fragments also form part of the
present invention. Preferred variants are those that vary from the
referents by conservative amino acid substitutions--i.e., those that
substitute a residue with another of like characteristics. Typical such
substitutions are among Ala, Val, Leu and Ile; among Ser and Thr, among
the acidic residues Asp and Glu; among Asn and Gln; and among the basic
residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly
preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are
substituted, deleted, or added in any combination.
The PIGR-1 polypeptides of the invention can be prepared in any suitable
manner. Such polypeptides include isolated naturally occurring
polypeptides, recombinantly produced polypeptides, synthetically produced
polypeptides, or polypeptides produced by a combination of these methods.
Means for preparing such polypeptides are well understood in the art.
Polynucleotides of the Invention
Another aspect of the invention relates to PIGR-1 polynucleotides. PIGR-1
polynucleotides include isolated polynucleotides which encode the PIGR-1
polypeptides and fragments, and polynucleotides closely related thereto.
More specifically, PIGR-1 polynucleotide of the invention include a
polynucleotide comprising the nucleotide sequence contained in SEQ ID NO:1
encoding a PIGR-1 polypeptide of SEQ ID NO: 2, and polynucleotides having
the particular sequences of SEQ ID NOS:1 and 3. PIGR-1 polynucleotides
further include a polynucleotide comprising a nucleotide sequence that has
at least 80% identity over its entire length to a nucleotide sequence
encoding the PIGR-1 polypeptide of SEQ ID NO: 2, and a polynucleotide
comprising a nucleotide sequence that is at least 80% identical to that of
SEQ ID NO:1 over its entire length. In this regard, polynucleotides at
least 90% identical are particularly preferred, and those with at least
95% are especially preferred Furthermore, those with at least 97% are
highly preferred and those with at least 98-99% are most highly preferred,
with at least 99% being the most preferred. Also included under PIGR-1
polynucleotides are a nucleotide sequence which has sufficient identity to
a nucleotide sequence contained in SEQ ID NO:1 to hybridize under
conditions useable for amplification or for use as a probe or marker. The
invention also provides polynucleotides which are complementary to such
PIGR-1 polynucleotides.
PIGR-1 of the invention is structurally related to other proteins of the
Immunoglobulin superfamily, as shown by the results of sequencing the cDNA
encoding human PIGR-1. The cDNA sequence of SEQ ID NO:1 contains an open
reading frame (nucleotide number 132 to 734) encoding a polypeptide of 201
amino acids of SEQ ID NO:2. The amino acid sequence of Table 2 (SEQ ID
NO:2) has about 42.65% identity (using BLASTX) in 67 amino acid residues
with CMRF35 (D. G. Jackson et al., Eur. J. Immunol. 22:1157-1163, 1992).
Furthermore, PIGR-1 (SEQ ID NO:2) is 30% identical to the poly-Ig receptor
over 90 amino acid residues (P. Krajci et al., Biochem. Biophys. Res.
Commun. 158:783-789, 1989). The nucleotide sequence of Table 1 (SEQ ID
NO:1) has about 68.64% identity (using BLASTN) in 118 nucleotide residues
with Human CMRF35 mRNA (D. G. Jackson et al., Eur. J. Immunol.
22:1157-1163, 1992). Thus, PIGR-1 polypeptides and polynucleotides of the
present invention are expected to have, inter alia, similar biological
functions/properties to their homologous polypeptides and polynucleotides,
and their utility is obvious to anyone skilled in the art.
TABLE 1.sup.a
1 CCGGGTCGAC CCACGCGTCC GTGTGCAGAA GGTGCAAGCC AGAGCTCAGG
51 CAGAACTTCC AGAGTGCATC TGGGATCTGC ATTTGCCACT GGTTGCAGAT
101 CAGGCGGACG AGGAGCCGGG AAGGCAGAGC CATGTGGCTG CCCCCTGCTC
151 TGCTCCTTCT CAGCCTCTCA GGCTGTTTCT CCATCCAAGG CCCAGAGTCT
201 GTGAGAGCCC CAGAGCAGGG GTCCCTGACG GTTCAATGCC ACTATAAGCA
251 AGGATGGGAG ACCTACATTA AGTGGTGGTG CCGAGGGGTG CGCTGGGATA
301 CATGCAAGAT CCTCATTGAA ACCAGAGGGT CGGAGCAAGG AGAGAAGAGT
351 GACCGTGTGT CCATCAAGGA CAATCAGAAA GACCGCACGT TCACTGTGAC
401 CATGGAGGGG CTCAGGCGAG ATGACGCAGA TGTTTACTGG TGTGGGATTG
451 AAAGAAGAGG ACCTGACCTT GGGACTCAAG TGAAAGTGAT TGTTGACCCA
501 GAGGGAGCGG CTTCCACAAC AGCAAGCTCA CCTACCAACA GCAATATGGC
551 AGTGTTCATC GGCTCCCACA AGAGGAACCA CTACATGCTC CTGGTATTTG
601 TGAAGGTGCC CATCTTGCTC ATCTTGGTCA CTGCCATCCT CTGGTTGAAG
651 GGGTCTCAGA GGGTCCCTGA GGAGCCAGGG GAACAGCCTA TCTACATGAA
701 CTTCTCCGAA CCTCTGACTA AAGACATGGC CACTTAGAGA GATGGATCTG
751 CAGAGCCTTC CTGCCCTGGC CACGTTTCCA GAAGAGACTC GGGCTGTGGA
801 AGGAACATCT ACGAGTCCTC GGGATGCAGT GACTGAGATA GGGGCCCTGG
851 GCCTCCGCCC TGGCCTTGGA GCTGGTGGGC ACCTCCCTGT TCTGCACAGC
901 TCAGGGACTT AGCCAGGTCC TCTCCTGAGC CACCATCACC TCCTGGGGTG
951 CCAGCACCTG TTCTCTTGGT CAGGAGCTGT AGAGATGGAG CTCAAGCACT
1001 GGACGACTCT GTCCCCACTG CTGGAATAAC TCGGGCACAG AGCATGGGAC
1051 CAAAGTACAG AAAGAGGTTG GGGGAGACCC CCCCAGCCCT AGACTTCCAT
1101 CATTCCGGAG ACCAACTCAA CACCGTCTTT GCCTGAGAAC CTGATATATC
1151 CGTGTTTTTA AATTTTTTTT TTTCTAGCAA AGTTGGGTTT TAATGACTTA
1201 TGTTCATAGG AAACCTCTCT GATCCCACAC ACAAGGAGGG TGATTCTGGG
1251 ATGAGTTCCT GGTTCTAGGG CATGAGGGGC TGGATGGACC CTGTCCCCAG
1301 GGAGGACATG GCTCTGAGTC CACAGGGCTG AGGAGGCAAT GGGAACCTCC
1351 CTGGCCCGGC CCGGTGGTTG GCCTCCCCCT CCCACCTCTT CCTCCTCCTA
1401 GCTCCCCAAG CTCCCTGCCT ATTCCCCCAC CTCCGAGGGG CTGCAGCTTG
1451 GGAGCCTCCT CAGCATGACA GCTTGGGTCT CCTCCCCAAA AGAGCCTGTC
1501 AGGCCTCAAG AACCACCTCC AGGTGGGGAG GGCAGTAACG AAAACCATCG
1551 CAGGAAATGG CACCCTCCCT TTTCGGTGAT GTTGAAATCA TGTTACTAAT
1601 GAAAACTGTC CTAGGGAAGT GGTTCTGTCT CCTCACAGGC TTCACCCACG
1651 GCGATGAGGC CCTTGAATGT GGTCACTTTG TGCTGTATGG TTGAGGGACC
1701 CTCACACCAA AGGGACCTTC CCATGTGAGA TGTGCTCCCG CCCCCACCTG
1751 CCCACAAGCA AACACACCAC ACATGTTCGG CATGTTGCCG TTTGAACACC
1801 CATGAGGACG CCTCCAACCT GCTCTTGGTT CTAATAGGGA GTACTGACTG
1851 TCAGCAGTGG ATAAAGGAGA GGGGACCCTC TGGTCCCTAG CATGGCACCC
1901 AGAGCCTCCC CTCTTCTTGT CCTTCAGCCA AAGAGAAACT TTCTCTGACT
1951 TTGAACTGAA TTTAGGTCTC TGGCCAATGA TGGGCCTGAA AATTCCATAA
2001 TGGCCAGAGA GGAGAGTTCG AGCCCGGCTA AGATCCCCTG AGTCATTCTG
2051 TGAGGGACCA AGACCCACAG TCCACCAGCC CCAGGGCCCT ACCTCCTGGA
2101 ATGCTTTCCT GGATCCAGCT TCCCGAAGAT CCGACCAGAC CCAGGGAGGA
2151 CGGCACCGCT CCGCGGGAGG GAAAGCCAAA GCATGGTGCT TCACCAGCTG
2201 GACTCAGGGG CGAGGGGACA TGGGCGCTTG TCAACGTGAT GTCATTCTTT
2251 TCCCACCGTT TCTTCCTGTT GATATTCAAT GAATCCGTCA ATCTCTCTGG
2301 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAA
.sup.a nucleotide sequence of a human PIGR-1 (SEQ ID NO:1).
TABLE 2.sup.b
1 MWLPPALLLL SLSGCFSIQG PESVRAPEQG SLTVQCHYKQ GWETYIKWWC
51 RGVRWDTCKI LIETRGSEQG EKSDRVSIKD NQKDRTFTVT MEGLRRDDAD
101 VYWCGIERRG PDLGTQVKVI VDPEGAASTT ASSPTNSNMA VFIGSHKRNH
151 YMLLVFVKVP ILLILVTAIL WLKGSQRVPE EPGEQPIYMN FSEPLTKDMA
201 T
.sup.b amino acid sequence of a human PIGR-1 (SEQ ID NO:2).
One polynucleotide of the present invention encoding PIGR-1 may be obtained
using standard cloning and screening, from a cDNA library derived from
mRNA in cells of human bone marrow, macrophage, eosinophil, activated
neutrophils and T cells using the expressed sequence tag (EST) analysis
(Adams, M. D. et al., Science (1991) 252:1651-1656; Adams, M. D. et al.,
Nature, (1992) 355:632-634; Adams M. D., et al., Nature (1995) 377
Supp:3-174). Polynucleotides of the invention can also be obtained from
natural sources such as genomic DNA libraries or can be synthesized using
well commercially available techniques.
The nucleotide sequence encoding PIGR-1 polypeptide of SEQ ID NO:2 may be
identical to the polypeptide encoding sequence contained in Table 1
(nucleotide number 132 to 734 of SEQ ID NO:1), or it may be a sequence,
which as a result of the redundancy (degeneracy) of the genetic code, also
encodes the polypeptide of SEQ ID NO:2.
When the polynucleotides of the invention are used for the recombinant
production of PIGR-1 polypeptide, the polynucleotide may include the
coding sequence for the mature polypeptide or a fragment thereof, by
itself; the coding sequence for the mature polypeptide or fragment in
reading frame with other coding, sequences, such as those encoding a
leader or secretory sequence, a pre-, or pro- or prepro-protein sequence,
or other fusion peptide portions. For example, a marker sequence which
facilitates purification of the fused polypeptide can be encoded. In
certain preferred embodiments of this aspect of the invention, the marker
sequence is a hexa-histidine peptide, as provided in the pQE vector
(Qiagen, Inc.) and described in Gentz et al., Proc Natl Acad Sci USA
(1989) 86:821-824, or is an HA tag. The polynucleotide may also contain
non-coding 5' and 3' sequences, such as transcribed, non-translated
sequences, splicing and polyadenylation signals, ribosome binding sites
and sequences that stabilize mRNA.
Further preferred embodiments are polynucleotides encoding PIGR-1 variants
comprising the amino acid sequence of PIGR-1 polypeptide of Table 2 (SEQ
ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues
are substituted, deleted or added, in any combination. Among the preferred
polynucleotides of the present invention is contained in Table 3 (SEQ ID
NO: 3) encoding the amino acid sequence of Table 4 (SEQ ID NO: 4).
TABLE 3.sup.c
1 TGTGCAGAAG GTGCAAGCCA GAGCTCAGGC AGAACTTCCA GAGTGCATCT
51 GGGATCTGCA TTTGCCACTG GTTGCAGATC AGGCGGACGA GGAGCCGGGA
101 AGGCAGAGCC ATGTGGCTGC CCCCTGCTCT GCTCCTTCTC AGCCTCTCAG
151 GCTGTTTCTC CATCCAAGGC CCAGAGTCTG TGAGAGCCCC AGAGCAGGGG
201 TCCCTGACGG TTCAATGCCA CTATAAGCAA GGATGGGAGA CCTACATTAA
251 GTGGTGGTGC CGAGGGGTGC GCTGGGATAC ATGCAAGATC CTCATTGAAA
301 CCAGAGGGTC GGAGCAAGGA GAGAAGAGTG ACCGTGTGTC CATCAAGGAC
351 AATCAGAAAG ACCGCACGTT CACTGTGACC ATGGAGGGGC TCAGGCGAGA
401 TGACGCAGAT GTTTACTGGT GTGGGATTGA AAGAAGAGGA CCTGACCTTG
451 GGACTCAAGT GAAAATTGAT TGTTNACCCA GAGGGAGCGG CTTTCCACAA
501 CAGCAAAGCT CACCTACCAA CAGCAATATG GCAGTGTTCA TCGGCTCCCA
551 CAAGAGGAAC CACTACATGC TCCTGGTATT TGTGAAGGTG CCCATCTTGC
601 TCATCTTGGT CAATGCCATN CTCTGGTTGA AAGGGTCTCA GAGGGTCCCT
651 GAGGAGCCAN GGGAACAGCC TATCTACATG GACTTCTCCG GACTCTGACT
701 AAAGACAT
.sup.c A partial nucleotide sequence of a human PIGR-1 (SEQ ID NO:3).
TABLE 4.sup.d
1 MWLPPALLLL SLSGCFSIQG PESVRAPEQG SLTVQCHYKQ GWETYIKWWC
51 RGVRWDTCKI LIETRGSEQG EKSDRVSIKD NQKDRTFTVT MEGLRRDDAD
101 VYWCGIERRG PDLGTQVKID CXPRGSGFPQ QQSSPTNSNM AVFIGSHKRN
151 HYMLLVFVKV PILLILVNAX LWLKGSQRVP EEPXEQPIYM DFSGL
.sup.d A partial amino acid sequence of a human PIGR-1 (SEQ ID NO:4).
The present invention further relates to polynucleotides that hybridize to
the herein above-described sequences. In this regard, the present
invention especially relates to polynucleotides which hybridize under
stringent conditions to the herein above-described polynucleotides. As
herein used, the term "stringent conditions" means hybridization will
occur only if there is at least 80%, and preferably at least 900%, and
more preferably at least 95%, yet even more preferably 97-99% identity
between the sequences.
Polynucleotides of the invention, which are identical or sufficiently
identical to a nucleotide sequence contained in SEQ ID NO:1 or a fragment
thereof, may be used as hybridization probes for cDNA and genomic DNA, to
isolate full-length cDNAs and genomic clones encoding PIGR-1 and to
isolate cDNA and genomic clones of other genes (including genes encoding
homologs and orthologs from species other than human) that have a high
sequence similarity to the PIGR-1 gene. Such hybridization techniques are
known to those of skill in the art. Typically these nucleotide sequences
are 80% identical, preferably 90% identical, more preferably 95% identical
to that of the referent. The probes generally will comprise at least 15
nucleotides. Preferably, such probes will have at least 30 nucleotides and
may have at least 50 nucleotides. Particularly preferred probes will range
between 30 and 50 nucleotides.
In one embodiment, to obtain a polynucleotide encoding PIGR-1 polypeptide,
including homologs and orthologs from species other than human, comprises
the steps of screening an appropriate library under stingent hybridization
conditions with a labeled probe having the SEQ ID NO: 1 or a fragment
thereof (including that of SEQ ID NO: 3), and isolating full-length cDNA
and genomic clones containing said polynucleotide sequence. Such
hybridization techniques are well known to those of skill in the art.
Stringent hybridization conditions are as defined above or alternatively
conditions under overnight incubation at 42.degree. C. in a solution
comprising: 50% formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium
citrate), 50 mM sodium phosphate (pH7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA,
followed by washing the filters in 0.1.times.SSC at about 65.degree. C.
The polynucleotides and polypeptides of the present invention may be
employed as research reagents and materials for discovery of treatments
and diagnostics to animal and human disease.
Vectors, Host Cells, Expression
The present invention also relates to vectors which comprise a
polynucleotide or polynucleotides of the present invention, and host cells
which are genetically engineered with vectors of the invention and to the
production of polypeptides of the invention by recombinant techniques.
Cell-free translation systems can also be employed to produce such
proteins using RNAs derived from the DNA constructs of the present
invention.
For recombinant production, host cells can be genetically engineered to
incorporate expression systems or portions thereof for polynucleotides of
the present invention. Introduction of polynucleotides into host cells can
be effected by methods described in many standard laboratory manuals, such
as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et
al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium
phosphate transfection, DEAE-dextran mediated transfection, transvection,
microinjection, cationic lipid-mediated transfection, electroporation,
transduction, scrape loading, ballistic introduction or infection.
Representative examples of appropriate hosts include bacterial cells, such
as streptococci, staphylococci, E. coli, Streptomyces and Bacillus
subtilis cells; fungal cells, such as yeast cells and Aspergillus cells;
insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells
such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells;
and plant cells.
A great variety of expression systems can be used. Such systems include,
among others, chromosomal, episomal and virus-derived systems, e.g.,
vectors derived from bacterial plasmids, from bacteriophage, from
transposons, from yeast episomes, from insertion elements, from yeast
chromosomal elements, from viruses such as baculoviruses, papova viruses,
such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,
pseudorabies viruses and retroviruses, and vectors derived from
combinations thereof, such as those derived from plasmid and bacteriophage
genetic elements, such as cosmids and phagemids. The expression systems
may contain control regions that regulate as well as engender expression.
Generally, any system or vector suitable to maintain, propagate or express
polynucleotides to produce a polypeptide in a host may be used. The
appropriate nucleotide sequence may be inserted into an expression system
by any of a variety of well-known and routine techniques, such as, for
example, those set forth in Sambrook et al., MOLECULAR CLONING, A
LABORATORY MANUAL (supra).
For secretion of the translated protein into the lumen of the endoplasmic
reticulum, into the periplasmic space or into the extracellular
environment, appropriate secretion signals may be incorporated into the
desired polypeptide. These signals may be endogenous to the polypeptide or
they may be heterologous signals.
If the PIGR-1 polypeptide is to be expressed for use in screening assays,
generally, it is preferred that the polypeptide be produced at the surface
of the cell. In this event, the cells may be harvested prior to use in the
screening assay. If PIGR-1 polypeptide is secreted into the medium, the
medium can be recovered in order to recover and purify the polypeptide; if
produced intracellularly, the cells must first be lysed before the
polypeptide is recovered.
PIGR-1 polypeptides can be recovered and purified from recombinant cell
cultures by well-known methods including ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity chromatography, hydroxylapatite chromatography and lectin
chromatography. Most preferably, high performance liquid chromatography is
employed for purification. Well known techniques for refolding proteins
may be employed to regenerate active conformation when the polypeptide is
denatured during isolation and or purification.
Diagnostic Assays
This invention also relates to the use of PIGR-1 polynucleotides for use as
diagnostic reagents. Detection of a mutated form of PIGR-1 gene associated
with a dysfunction will provide a diagnostic tool that can add to or
define a diagnosis of a disease or susceptibility to a disease which
results from under-expression, over-expression or altered expression of
PIGR-1. Individuals carrying mutations m the PIGR-1 gene may be detected
at the DNA level by a variety of techniques.
Nucleic acids for diagnosis may be obtained from a subject's cells, such as
from blood, urine, saliva, tissue biopsy or autopsy material. The genomic
DNA may be used directly for detection or may be amplified enzymatically
by using PCR or other amplification techniques prior to analysis. RNA or
cDNA may also be used in similar fashion. Deletions and insertions can be
detected by a change in size of the amplified product in comparison to the
normal genotype. Point mutations can be identified by hybridizing
amplified DNA to labeled PIGR-1 nucleotide sequences. Perfectly matched
sequences can be distinguished from mismatched duplexes by RNase digestion
or by differences in melting temperatures. DNA sequence differences may
also be detected by alterations in electrophoretic mobility of DNA
fragments in gels, with or without denaturing agents, or by direct DNA
sequencing. See, e.g., Myers et al., Science (1985) 230:1242. Sequence
changes at specific locations may also be revealed by nuclease protection
assays, such as RNase and S1 protection or the chemical cleavage method.
See Cotton et al., Proc Natl Acad Sci USA (1985) 85: 4397-4401. In another
embodiment, an array of oligonucleotides probes comprising PIGR-1
nucleotide sequence or fragments thereof can be constructed to conduct
efficient screening of e.g., genetic mutations. Array technology methods
are well known and have general applicability and can be used to address a
variety of questions in molecular genetics including gene expression,
genetic linkage, and genetic variability. (See for example: M. Chee et
al., Science, Vol 274, pp 610-613 (1996)).
The diagnostic assays offer a process for diagnosing or determining a
susceptibility to rheumatoid arthritis (RA), multiple sclerosis (MS),
psoriasis, systemic lupus erythematosus (SLE) and imflammatory bowel
disease (IBD) through detection of mutation in the PIGR-1 gene by the
methods described
In addition, rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis,
systemic lupus erythematosus (SLE) and inflammatory bowel disease (IBD),
can be diagnosed by methods comprising determining from a sample derived
from a subject an abnormally decreased or increased level of PIGR-1
polypeptide or PIGR-1 mRNA. Decreased or increased expression can be
measured at the RNA level using any of the methods well known in the art
for the quantitation of polynucleotides, such as, for example, PCR,
RT-PCR, RNase protection, Northern blotting and other hybridization
methods. Assay techniques that can be used to determine levels of a
protein, such as an PIGR-1, in a sample derived from a host are well-known
to those of skill in the art. Such assay methods include
radioimmunoassays, competitive-binding assays, Western Blot analysis and
ELISA assays.
Thus in another aspect, the present invention relates to a diagonostic kit
for a disease or suspectability to a disease, particularly rheumatoid
arthritis (RA), multiple sclerosis (MS), psoriasis, systemic lupus
erythematosus (SLE) and inflammatory bowel disease (IBD), which comprises:
(a) a PIGR-1 polynucleotide, preferably the nucleotide sequence of SEQ ID
NO: 1, or a fragment thereof;
(b) a nucleotide sequence complementary to that of (a);
(c) a PIGR-1 polypeptide, preferably the polypeptide of SEQ ID NO: 2, or a
fragment thereof; or
(d) an antibody to a PIGR-1 polypeptide, preferably to the polypeptide of
SEQ ID NO: 2.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may
comprise a substantial component.
Chromosome Assays
The nucleotide sequences of the present invention are also valuable for
chromosome identification. The sequence is specifically targeted to and
can hybridize with a particular location on an individual human
chromosome. The mapping of relevant sequences to chromosomes according to
the present invention is an important first step in correlating those
sequences with gene associated disease. Once a sequence has been mapped to
a precise chromosomal location, the physical position of the sequence on
the chromosome can be correlated with genetic map data. Such data are
found, for example, in V. McKusick, Mendelian Inheritance in Man
(available on line through Johns Hopkins University Welch Medical
Library). The relationship between genes and diseases that have been
mapped to the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes). The differences in
the cDNA or genomic sequence between affected and unaffected individuals
can also be determined. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the mutation
is likely to be the causative agent of the disease.
Antibodies
The polypeptides of the invention or their fragments or analogs thereof, or
cells expressing them can also be used as immunogens to produce antibodies
immunospecific for the PIGR-1 polypeptides. The term "immunospecific"
means that the antibodies have substantiall greater affinity for the
polypeptides of the invention than their affinity for other related
polypeptides in the prior art.
Antibodies generated against the PIGR-1 polypeptides can be obtained by
administering the polypeptides or epitope-bearing fragments, analogs or
cells to an animal, preferably a nonhuman, using routine protocols. For
preparation of monoclonal antibodies, any technique which provides
antibodies produced by continuous cell line cultures can be used. Examples
include the hybridoma technique (Kohler, G. and Milstein, C., Nature
(1975) 256:495-497), the trioma technique, the human B-cell hybridoma
technique (Kozbor et al., Immunology Today (1983) 4:72) and the
EBV-hybridoma technique (Cole et al., MONOCLONAL ANTIBODIES AND CANCER
THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).
Techniques for the production of single chain antibodies (U.S. Pat. No.
4,946,778) can also be adapted to produce single chain antibodies to
polypeptides of this invention. Also, transgenic mice, or other organisms
including other mammals, may be used to express humanized antibodies.
The above-described antibodies may be employed to isolate or to identify
clones expressing the polypeptide or to purify the polypeptides by
affinity chromatography.
Antibodies against PIGR-1 polypeptides may also be employed to treat
rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, systemic
lupus erythematosus (SLE) and inflammatory bowel disease (IBD), among
others.
Vaccines
Another aspect of the invention relates to a method for inducing an
immunological response in a mammal which comprises inoculating the mammal
with PIGR-1 polypeptide, or a fragment thereof, adequate to produce
antibody and/or T cell immune response to protect said animal from
rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, systemic
lupus erythematosus (SLE) and inflammatory bowel disease (IBD), among
others. Yet another aspect of the invention relates to a method of
inducing immunological response in a mammal which comprises, delivering
PIGR-1 polypeptide via a vector directing expression of PIGR-1
polynucleotide in vivo in order to induce such an immunological response
to produce antibody to protect said animal from diseases.
Further aspect of the invention relates to an immunological/vaccine
formulation (composition) which, when introduced into a mammalian host,
induces an immunological response in that mammal to a PIGR-1 polypeptide
wherein the composition comprises a PIGR-1 polypeptide or PIGR-1 gene. The
vaccine formulation may further comprise a suitable carrier. Since PIGR-1
polypeptide may be broken down in the stomach, it is preferably
administered parenterally (including subcutaneous, intramuscular,
intravenous, intradermal etc. injection). Formulations suitable for
parenteral administration include aqueous and non-aqueous sterile
injection solutions which may contain anti-oxidants, buffers,
bacteriostats and solutes which render the formulation isotonic with the
blood of the recipient; and aqueous and non-aqueous sterile suspensions
which may include suspending agents or thickening agents. The formulations
may be presented in unit-dose or multi-dose containers, for example,
sealed ampoules and vials and may be stored in a freeze-dried condition
requiring only the addition of the sterile liquid carrier immediately
prior to use. The vaccine formulation may also include adjuvant systems
for enhancing the immunogenicity of the formulation, such as oil-in water
systems and other systems known in the art. The dosage will depend on the
specific activity of the vaccine and can be readily determined by routine
experimentation.
Screening Assays
The PIGR-1 polypeptide of the present invention may be employed in a
screening process for compounds which bind the receptor and which activate
(agonists) or inhibit activation of (antagonists) the receptor polypeptide
of the present invention. Thus, polypeptides of the invention may also be
used to assess the binding of small molecule substrates and ligands in,
for example, cells, cell-free preparations, chemical libraries, and
natural product mixtures. These substrates and ligands may be natural
substrates and ligands or may be structural or functional mimetics. See
Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991).
PIGR-1 polypeptides are responsible for many biological functions,
including many pathologies. Accordingly, it is desirous to find compounds
and drugs which stimulate PIGR-1 on the one hand and which can inhibit the
function of PIGR-1 on the other hand. In general, agonists are employed
for therapeutic and prophylactic purposes for such conditions as
rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, systemic
lupus erythematosus (SLE) and inflammatory bowel disease (IBD) Antagonists
may be employed for a variety of therapeutic and prophylactic purposes for
such conditions as rheumatoid arthritis (RA), multiple sclerosis (MS),
psoriasis, systemic lupus erythematosus (SLE) and inflammatory bowel
disease (IBD).
In general, such screening procedures involve producing appropriate cells
which express the receptor polypeptide of the present invention on the
surface thereof. Such cells include cells from mammals, yeast, Drosophila
or E. coli. Cells expressing the receptor (or cell membrane containing the
expressed receptor) are then contacted with a test compound to observe
binding, or stimulation or inhibition of a functional response.
The assays may simply test binding of a candidate compound wherein
adherence to the cells bearing the receptor is detected by means of a
label directly or indirectly associated with the candidate compound or in
an assay involving competition with a labeled competitor. Further, these
assays may test whether the candidate compound results in a signal
generated by activation of the receptor, using detection systems
appropriate to the cells bearing the receptor at their surfaces.
Inhibitors of activation are generally assayed in the presence of a known
agonist and the effect on activation by the agonist by the presence of the
candidate compound is observed.
Further, the assays may simply comprise the steps of mixing a candidate
compound with a solution containing a PIGR-1 polypeptide to form a
mixture, measuring PIGR-1 activity in the mixture, and comparing the
PIGR-1 activity of the mixture to a standard.
The PIGR-1 cDNA, protein and antibodies to the protein may also be used to
configure assays for detecting the effect of added compounds on the
production of PIGR-1 mRNA and protein in cells. For example, an ELISA may
be constructed for measuring secreted or cell associated levels of PIGR-1
protein using monoclonal and polyclonal antibodies by standard methods
known in the art, and this can be used to discover agents which may
inhibit or enhance the production of PIGR-1 (also called antagonist or
agonist, respectively) from suitably manipulated cells or tissues.
Standard methods for conducting screening assays are well understood in
the art.
Examples of potential PIGR-1 antagonists include antibodies or, in some
cases, oligonucleotides or proteins which are closely related to the
ligand of the PIGR-1, e.g., a fragment of the ligand, or small molecules
which bind to the receptor but do not elicit a response, so that the
activity of the receptor is prevented.
Thus in another aspect, the present invention relates to a screening kit
for identifying agonists, antagonists, ligands, receptors, substrates,
enzymes, etc. for PIGR-1 polypeptides; or compounds which decrease or
enhance the production of PIGR-1 polypeptides, which comprises:
(a) a PIGR-1 polypeptide, preferably that of SEQ ID NO:2;
(b) a recombinant cell expressing a PIGR-1 polypeptide, preferably that of
SEQ ID NO:2;
(c) a cell membrane expressing a PIGR-1 polypeptide; preferably that of SEQ
ID NO: 2; or
(d) antibody to a PIGR-1 polypeptide, preferably that of SEQ ID NO: 2.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may
comprise a substantial component.
Prophylactic and Therapeutic Methods
This invention provides methods of treating an abnormal conditions related
to both an excess of and insufficient amounts of PIGR-1 activity.
If the activity of PIGR-1 is in excess, several approaches are available.
One approach comprises administering to a subject an inhibitor compound
(antagonist) as hereinabove described along with a pharmaceutically
acceptable carrier in an amount effective to inhibit activation by
blocking binding of ligands to the PIGR-1, or by inhibiting a second
signal, and thereby alleviating the abnormal condition.
In another approach, soluble forms of PIGR-1 polypeptides still capable of
binding the ligand in competition with endogenous PIGR-1 may be
administered. Typical embodiments of such competitors comprise fragments
of the PIGR-1 polypeptide.
In still another approach, expression of the gene encoding endogenous
PIGR-1 can be inhibited using expression blocking techniques. Known such
techniques involve the use of antisense sequences, either internally
generated or separately administered. See, for example, O'Connor, J
Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors
of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Alternatively,
oligonucleotides which form triple helices with the gene can be supplied.
See, for example, Lee et al., Nucleic Acids Res (1979) 6:3073; Cooney et
al., Science (1988) 241:456; Dervan et al., Science (1991) 251:1360. These
oligomers can be administered per se or the relevant oligomers can be
expressed in vivo.
For treating abnormal conditions related to an under-expression of PIGR-1
and its activity, several approaches are also available. One approach
comprises administering to a subject a therapeutically effective amount of
a compound which activates PIGR-1, i.e., an agonist as described above, in
combination with a pharmaceutically acceptable carrier, to thereby
alleviate the abnormal condition. Alternatively, gene therapy may be
employed to effect the endogenous production of PIGR-1 by the relevant
cells in the subject. For example, a polynucleotide of the invention may
be engineered for expression in a replication defective retroviral vector,
as discussed above. The retroviral expression construct may then be
isolated and introduced into a packaging cell transduced with a retroviral
plasmid vector containing RNA encoding a polypeptide of the present
invention such that the packaging cell now produces infectious viral
particles containing the gene of interest. These producer cells may be
administered to a subject for engineering cells in vivo and expression of
the polypeptide in vivo. For overview of gene therapy, see Chapter 20,
Gene Therapy and other Molecular Genetic-based Therapeutic Approaches,
(and references cited therein) in Human Molecular Genetics, T Strachan and
A P Read, BIOS Scientific Publishers Ltd (1996).
Formulation and Administration
Peptides, such as the soluble form of PIGR-1 polypeptides, and agonists and
antagonist peptides or small molecules, may be formulated in combination
with a suitable pharmaceutical carrier. Such formulations comprise a
therapeutically effective amount of the polypeptide or compound, and a
pharmaceutically acceptable carrier or excipient. Such carriers include
but are not limited to, saline, buffered saline, dextrose, water,
glycerol, ethanol, and combinations thereof. Formulation should suit the
mode of administration, and is well within the skill of the art. The
invention further relates to pharmaceutical packs and kits comprising one
or more containers filled with one or more of the ingredients of the
aforementioned compositions of the invention.
Polypeptides and other compounds of the present invention may be employed
alone or in conjunction with other compounds, such as therapeutic
compounds.
Preferred forms of systemic administration of the pharmaceutical
compositions include injection, typically by intravenous injection. Other
injection routes, such as subcutaneous, intramuscular, or intraperitoneal,
can be used. Alternative means for systemic administration include
transmucosal and transdermal administration using penetrants such as bile
salts or fusidic acids or other detergents. In addition, if properly
formulated in enteric or encapsulated formulations, oral administration
may also be possible. Administration of these compounds may also be
topical and/or localized, in the form of salves, pastes, gels and the
like.
The dosage range required depends on the choice of peptide, the route of
administration, the nature of the formulation, the nature of the subject's
condition, and the judgment of the attending practitioner. Suitable
dosages, however, are in the range of 0.1-100 .mu.g/kg of subject. Wide
variations in the needed dosage, however, are to be expected in view of
the variety of compounds available and the differing efficiencies of
various routes of administration. For example, oral administration would
be expected to require higher dosages than administration on by
intravenous injection. Variations in these dosage levels can be adjusted
using standard empirical routines for optimization, as is well understood
in the art.
Polypeptides used in treatment can also be generated endogenously in the
subject, in treatment modalities often referred to as "gene therapy" as
described above. Thus, for example, cells from a subject may be engineered
with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex
vivo, and for example, by the use of a retroviral plasmid vector. The
cells are then introduced into the subject.
EXAMPLES
The examples below are carried out using standard techniques, which are
well known and routine to those of sill in the art, except where otherwise
described in detail. The examples illustrate, but do not limit the
invention.
Example 1
While there are several methods to obtain the full length cDNA, two are
outlined below.
1) The method of Rapid Amplification of cDNA Ends (RACE) can be utilized to
obtain the 5' end. See Frohman et al., Proc. Nat. Acad. Sci USA 85,
8998-9002. (1988). Briefly, specific oliognucleotides are annealed to mRNA
and used to prime the synthesis of the cDNA strand. Following destruction
of the mRNA with RNaseH, a poly C anchor sequence is added to the 3' end
of the cDNA and the resulting fragment is amplified using a nested set of
antisense primers and an anchor sequence primer. The amplified fragment is
cloned into an appropriate vector and subjected to restriction and
sequence analysis.
2) The polymerase chain reaction can be used to amplify the 5' end of the
cDNA from human cDNA libraries using sequential rounds of nested PCR with
two sets of primers. One set of antisense primers is specific to the 5'
end of the partial cDNA and the other set of primers anneals to a vector
specific sequence. The amplified products are cloned into an appropriate
vector and subjected to restriction and sequence analysis.
Example 2
PIGR-1 Belongs to Immunoglobulin (Ig) Superfamily
The extracellular region of PIGR-1 contains a single Ig domain with a
V-like fold as shown by (1) the presence of Ig V fold conserved residues
and (2) homology to several other Ig like proteins (poly Ig V1 and V4,
CMRF35, TCR V.beta. and Ig .kappa. V.sub.L).
In the following alignment, conserved Ig V residues are shown in bold and
residues in PIGR-1 shared with at least 3 of the other members are noted
with a *.
B C C' C"
PIGR-1 IQGPESVAAPEQGSLTVQCHYK+L QGWETYGVRWDTCKILI+L ETRGSEQ+L
GEKSDR+L 75
(AA 18-75 of
SEQ ID NO:2)
PolyIgRV1
IFGPEEVNSVEGNSVSITCYYPPTSVNTRKYWC.RQGARG..CITLISSEGYVSSKYAGR 57
(SEQ ID NO:5)
PolyIgRV4
PRSPTVVKGVAGSSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLLVDSEGWVKAQYEGR 60
(SEQ ID NO:6)
CMRF35
LSHPMTVAGPVGGSLSVQCRYEKEHRTLNKFWC..RPPQILRCDKIVETKGS.AGKRNGR 56
(SEQ ID NO:7)
TCR v.beta.
SQKPSRDICQRGTSLTIQCQV.DSQVTMMFWYRQQPGQSLTLIATANQGSEATYESGFVI 60
(SEQ ID NO:8)
Ig k V.sub.L
TQTPASVEVAVGGTVTIKCQASQSISTYLSWYQQKPGQRPKLLIY....RASTLASG.VS 56
(SEQ ID NO:9)
* * * * * * * ** * * *
*
D E F
PIGR-1 VSIKDNQKDRTF.TVTMEGLRRDDADVYWCGIERRGPDLGTQVKVIV
(AA 76-121 of
SEQ ID NO:2)
PolyIgRV1 ANLTNFPENGTF.TVILNQLSQDDSGRYKCGLGINSRGLSFDVSLEV
(SEQ ID NO:10)
PolyIgV4 LSLLEEPGNGTF.TVILNQLTSRDAGFYWC...LTNGDTLWRTTVEI
(SEQ ID NO:11)
CMRF35 VSIRDSPANLSF.TVTLENLTEEDAGTYWCGVDTPWLRDFHDPIVEV
(SEQ ID NO:12)
TCR v.beta. DKFPISRPNLTFSTLTVSNMSPEDSSIYLCSVE..GEAGDTQY.FGP
(SEQ ID NO:13)
Ig k V.sub.L SRFKGSGSGTEF.TLTISGVECADAATYYCQQGWSSSNVEN...VFG
(SEQ ID NO:14)
** *** * ** * * *
Example 3
PIGR-1 Gene Expression Pattern
PIGR-1, a new member of the Ig superfamily, has been identified. The
predicted protein sequence of this new gene shows modest, but extended,
homology to CMRF-35, particularly in the extracellular domain. Based on
the library source of the EST sequences comprising PIGR-1 which were
isolated from leucocytes such as macrophage, neutrophil, eosinophil and T
cells, its expression is restricted to leucocytes, suggesting a role in
immune function. Thus, this protein is a candidate target for diseases of
the immune system such as rheumatoid arthritis (RA), multiple sclerosis
(MS), psoriasis, systemic lupus erythematosus (SLE) and inflammatory bowel
disease (IBD).
Example 4
Recombinant Soluble PIGR-1 Proteins
The extracellular domain of PIGR-1 is expressed as a secreted soluble
protein by truncation at the start of the transmembrane domain (asparagine
149 or histidine 150 in Table 2) as has been described for other
immunoglobulin domain proteins, e.g. for CD4 (K. C. Deen et al., Nature
331: 82-84 (1988)). PIGR-1 is also expressed as a secreted, soluble Ig
fusion protein by linking the same extracellular region of PIGR-1 to the
hinge and constant domains of heavy chain IgG such as has been described
for CD4 (D. J. Capon et al., Nature 317: 525-531 (1989)). In addition,
preparation of oligomeric Ig fusion proteins is possible by addition of
the tailpiece segment of IgM or IgA to the C-terminus of the Fc domain of
IgGs, as exemplified for the IgM tailpiece segment in R. I. F. Smith and
S. L. Morrison, Biotechnology 12: 683-688 (1994) and in R. I. F. Smith, et
al., J. Immunol. 154: 2226-2236 (1995). These proteins are produced in
insect cells or in mammalian cells such as COS-7 or CHO, purified by
standard methodology, and are useful as tool, therapeutic, and diagnostic
agents. Thus, these proteins are used to:
a) Determine the cleavage site of the N-terminal leader by amino acid
sequence analysis of this processed recombinant protein.
b) Prepare polyclonal and monoclonal antibodies for:
1) Detection of PIGR-1 protein expression in different tissues and cell
types.
2) Functional studies of PIGR-1 protein, such as induction of cell
differentiation and proliferation, cytokine production, and cell death
assays.
c) Test for agonist/antagonist activity when added to cultured cells and in
animal models of immune disease.
d) Search for its ligand(s).
e) Establish screen assays for small molecule agonists or antagonists of
PIGR-1 protein, which may be potential therapeutic and/or diagnostic
agents.
All publications, including but not limited to patents and patent
applications, cited in this specification are herein incorporated by
reference as if each individual publication were specifically and
individually indicated to be incorporated by reference herein as though
fully set forth.
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